Morintides: cargo-free chitin-binding peptides from Moringa oleifera
© The Author(s). 2017
Received: 16 January 2017
Accepted: 17 March 2017
Published: 31 March 2017
Hevein-like peptides are a family of cysteine-rich and chitin-binding peptides consisting of 29–45 amino acids. Their chitin-binding property is essential for plant defense against fungi. Based on the number of cysteine residues in their sequences, they are divided into three sub-families: 6C-, 8C- and 10C-hevein-like peptides. All three subfamilies contain a three-domain precursor comprising a signal peptide, a mature hevein-like peptide and a C-terminal domain comprising a hinge region with protein cargo in 8C- and 10C-hevein-like peptides.
Here we report the isolation and characterization of two novel 8C-hevein-like peptides, designated morintides (mO1 and mO2), from the drumstick tree Moringa oleifera, a drought-resistant tree belonging to the Moringaceae family. Proteomic analysis revealed that morintides comprise 44 amino acid residues and are rich in cysteine, glycine and hydrophilic amino acid residues such as asparagine and glutamine. Morintides are resistant to thermal and enzymatic degradation, able to bind to chitin and inhibit the growth of phyto-pathogenic fungi. Transcriptomic analysis showed that they contain a three-domain precursor comprising an endoplasmic reticulum (ER) signal sequence, a mature peptide domain and a C-terminal domain. A striking feature distinguishing morintides from other 8C-hevein-like peptides is a short and protein-cargo-free C-terminal domain. Previously, a similar protein-cargo-free C-terminal domain has been observed only in ginkgotides, the 8C-hevein-like peptides from a gymnosperm Ginkgo biloba. Thus, morintides, with a cargo-free C-terminal domain, are a stand-alone class of 8C-hevein-like peptides from angiosperms.
Our results expand the existing library of hevein-like peptides and shed light on molecular diversity within the hevein-like peptide family. Our work also sheds light on the anti-fungal activity and stability of 8C-hevein-like peptides.
KeywordsCysteine-rich peptides Hevein Hevein-like peptides Moringa oleifera Anti-fungal Chitin-binding
Hevein, a 43-amino-acid-long-peptide comprising a conserved chitin-binding domain, was first isolated from the latex of the rubber tree Hevea brasiliensis by Archer et al. in 1960 . Subsequently, peptides homologous to hevein were isolated from different plants and named hevein-like peptides due to their similarity to hevein. Hevein and hevein-like peptides, are a family of cysteine-rich peptides (CRPs), 29–45 amino acids in length, and are rich glycine (5–7 residues). Based on the number of cysteine residues, they can be classified into three subfamilies, namely 6C-, 8C- and 10C-hevein-like peptides  (Additional file 1: Table S1). Hevein-like peptides play a role in plant defense by binding to chitin, a polymer of repeating N-acetylglucosamine (GlcNAc) units and a major constituent of fungal cell walls . Binding of hevein-like peptides to chitin is facilitated by a highly conserved domain called the hevein domain  or chitin-binding domain , which is composed of glycine, cysteine and aromatic residues stabilized by three to five disulfide bonds. The hevein domain is also found in chitin hydrolyzing enzymes called chitinases and Urtica dioica agglutinin (UDA) from stinging nettle Urtica dioica . Due to their chitin-binding property, hevein-like peptides inhibit the growth of a wide range of fungi .
Moringa oleifera belongs to the flowering plant family Moringaceae and is commonly known as the drumstick tree or the horse-radish tree [18, 19]. It was once native to the foothills of the Himalayas but is now cultivated globally, especially in tropical and sub-tropical areas [20, 21]. It is a drought-resistant tree that can grow to a height of 10 m, and its trunk can reach a diameter of 45 cm [22, 23]. M. oleifera leaves are a rich source of vitamin C, calcium, potassium, β-carotene and natural antioxidants . The stem-bark, flowers, seeds and roots are edible and nutritious, comprising compounds such as alkaloids, potassium, calcium, ascorbic acid and antioxidants [24, 25]. Since M. oleifera is rich in nutrients, it is commonly referred to as the “Miracle Tree” to combat malnutrition in developing countries . Due to its high medicinal and nutritional value, M. oleifera is now incorporated into health formulations that are marketed as remedies for a variety of health disorders . M. oleifera seeds are the best known natural coagulants and are commonly used to purify turbid water. The coagulation properties are likely due to dimeric proteins in the seeds that are highly stable and soluble in water . Aqueous and ethanol extracts of M. oleifera leaves are known to have anti-inflammatory, antimicrobial, anti-diabetic and anti-ulcer properties and cholesterol lowering and blood pressure stabilizing effects . The seeds, stem, roots and leaves of M. oleifera are reported to exhibit anti-fungal effects [18, 29]. However, there is no report on the peptide and protein components that contribute to the putative therapeutic value of M. oleifera leaves. Since hevein-like peptides are primarily involved in plant defense against fungi, we screened the leaves of M. oleifera for putative hevein-like peptides by mass spectrometric analysis.
In this study, we report two antifungal 8C-hevein-like peptides from M. oleifera, designated morintides and abbreviated as mO1 and mO2. Sequencing using LC-ESI-MS/MS and transcriptomic analysis showed that morintides mO1 and mO2 are 44 amino acids long. Transcriptome data (RNAseq) analysis revealed that the full-length precursors of morintides comprise, a signal peptide domain, a mature peptide domain and a short C-terminal domain. Interestingly, morintides have a protein-cargo-free C-terminal domain (15 residues) when compared to hevein (142 residues). Since mO1 was the most abundant peptide, its function and stability were characterized. As a consequence of its chitin-binding property, mO1 inhibited the growth of the fungal strains Alternaria alternata and Alternaria brassiciola, by inhibiting the growth of fungal hyphae. To the best of our knowledge, this is the first report of an 8C-hevein-like peptide from angiosperms without a C-terminal protein cargo. Together, our results provide insight into the sequence diversity of hevein-like peptides, with insights into their chitin-binding property, anti-fungal activity and high stability against proteolysis and thermal degradation.
General experimental procedures
Shimadzu systems were used for high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC). Phenomenex C18 columns (particle size, 5 μm; pore size, 300 Å; CA, USA) with dimensions of 250 × 22 mm, 250 × 10 mm, and 250 × 4.6 mm were used for preparative, semi-preparative, and analytical reverse-phase (RP)-HPLC, respectively. Strong cation exchange (SCX)-HPLC was performed on a polyLC polysulfoethyl A column (250 × 9.4 mm and 250 × 4.6 mm). Mass spectrometry (MS) analysis of crude extracts and HPLC fractions was performed using an ABI 4800 MALDI-TOF/TOF system (Applied Biosystems, Framingham, MA, USA). An Infinite® 200 PRO microplate reader (Tecan Group Ltd., Maennedorf, Switzerland Germany) was used to measure absorbance in chitin binding, cytotoxicity, and microbroth dilution assays. Chemical reagents of analytical grade were purchased from Sigma Aldrich (St. Louis, MO, USA) [30–33].
Isolation of peptides from M. oleifera
M. oleifera leaves (3 kg) were bought from a local market in Singapore, and were authenticated by an experienced herbalist Mr Ng Kim Chuan from Nanyang Herbs Garden (Nanyang Technological University, Singapore). A voucher specimen was deposited in the Nanyang Technological University Herbarium, School of Biological Sciences, Singapore. The fresh leaves were blended with an equal volume of water in an Oster Fusion Blender (Philips, Singapore) for 6 min. The homogenized plant material was then centrifuged at 8,000 rpm for 10 min at 4 °C in an Avanti J25 centrifuge (Beckman-Coulter, USA) [34, 35]. The supernatant was filtered and loaded onto a C18 flash column. Elution was performed using increasing concentrations of ethanol (20 70%). The presence of the desired peptides was confirmed by performing MALDI-TOF MS on the eluents. Fractions that contained the peptides of interest were then pooled and concentrated using a rotary evaporator at a pressure of 70 mbar at 42 °C. The concentrated eluents were then purified using multiple rounds of SCX and RP-HPLC. For SCX-HPLC a gradient from buffer A (20 mM NaH2PO4, 5% acetonitrile, pH 2.8) to buffer B (20 mM NaH2PO4, 5% acetonitrile, 0.5 M NaCl, pH 2.8) was used. Fractions from SCX-HPLC that contained the desired peptides were pooled and purified by RP-HPLC using a linear gradient from buffer A (0.1% trifluoroacetic acid in water) to buffer B (0.1% trifluoroacetic acid in 100% acetonitrile).
Extraction of RNA from fresh M. oleifera leaves was performed using TRIzol® Reagent (Life Technologies, Carlsbad, USA) following the manufacturer’s protocol. Briefly, fresh M. oleifera leaves (100 mg) were homogenized using liquid nitrogen and incubated with 1 ml of TRIzol® Reagent (Life Technologies, Carlsbad, USA) at room temperature for 5 min. Chloroform (200 μL) was added to the homogenized sample and incubated at room temperature for 3 min followed by centrifugation at 12,000 g for 15 min at 4 °C. The RNA-containing aqueous phase was incubated at room temperature for 10 min with an equal volume of isopropanol before centrifuging at 12,000 g for 10 min. The pellet containing RNA was washed twice with 75% ethanol (1 mL). The pellet was dried and resuspended in 30 μl of diethylpyrocarbonate (DEPC) water. The RNA sample was sent to Macrogen Inc. Korea for sequencing.
Sequence determination of morintides
Reduction and alkylation of morintides was performed as previously described [15, 36, 37]. Briefly, approximately 600 μg of purified peptide was incubated with 20 mM dithiothreitol (DTT) in 20 mM ammonium bicarbonate buffer (NH4HCO3, pH 7) at 37 °C for 2 h. The reduced peptide was then alkylated with 40 mM iodoacetamide at 37 °C for 1 h. The reduced-alkylated sample was desalted using Millipore ZipTips and lyophilized. The peptide was re-dissolved in 0.1% formic acid before mass spectrometry analysis. A Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific, Bremen, Germany) coupled with an Orbitrap Elite mass spectrometer (Thermo Scientific Inc., Bremen, Germany) was used to perform LC/MS-MS analysis. Elution was performed over a 60 min gradient from eluent A (0.1% formic acid) to eluent B (90% acetonitrile/0.1% formic acid). LTQ Tune Plus software (Thermo Fisher Scientific, Bremen, Germany) was used to set the mass spectrometer to positive mode for data acquisition. A Michrom’s Thermo CaptiveSpray nanoelectrospray ion source (Bruker-Michrom, Auburn, USA) was used to generate the spray. A Full FT-MS (350–2000 m/z, resolution 60.000, with 1 μscan per spectrum) was alternated with Full FT-MS and an FT-MS/MS scan applying 27%, 30 and 32% normalized collision energy in high-energy collisional dissociation (110–2000 m/z, resolution 30,000, with 2 μscan averaged per MS/MS spectrum) for data acquisition where three intense ions with a charge greater than +2 and a mass difference of 3 Da were isolated and fragmented. A source voltage of 1.5 kV and capillary temperature of 250 °C were used. Automatic gain control was set to 1 × 106 for full scan-MS and MS/MS. PEAKS studio (version 7.5, Bioinformatics Solutions, Waterloo, Canada) was used to process data from LC-MS/MS analysis with parent error tolerance and a fragment error tolerance of 10 ppm and 0.05 Da, respectively.
The precursor sequences were aligned using MUSCLE  and analyzed using neighbor joining agglomerative clustering by MEGA 6.0 . The phylogenetic tree was constructed using bootstrap method with 1000 replications and poisson model was employed for substitution modelling. The phylogenetic tree was displayed using iTOL .
Chitin binding assay
Purified morintide (mO1) was incubated with chitin beads (New England Biolabs, Massachusetts, UK) in chitin binding buffer (140 mM NaCl, 10 mM Tris EDTA 1 mM; pH 8.0; then, 0.1% (v/v) Tween 20 was added) at 4 °C for 4 h. At each time point, the beads were centrifuged at 12,000 g for 1 min and the absorbance of the supernatant was read at 280 nm to assess binding. Elution of the bound peptide was performed using two methods. In the first method, 0.5 M acetic acid and heating at 100 °C for 30 min and 1 h was used, and in the second method, four elution buffers (10 mM Tris, 1 mM EDTA, 0.1% (v/v) Tween 20; pH 8.0) with increasing concentration of sodium chloride (300 mM, 500 mM, 700 mM, and 1 M) were used. Reduced and alkylated mO1 was used as a control. Supernatants were further analyzed by UPLC and MALDI-TOF to assess binding and elution.
Disc diffusion assay
The susceptibility of seven fungal strains obtained from the China Center of Industrial Culture Collection (CICC), namely, Curvularia lunata (CICC 40301), Fusarium oxysporum (CICC 2532), Aspergillus niger (CICC 2089), Verticillium dahilae (CICC 2534), Rhizoctonia solani (CICC 40259), Alternaria alternata (CICC 2465) and Alternaria brassiciola (CICC 2646) to mO1 (17.5 to 70 μg) was tested using a disc diffusion assay . Potato dextrose agar (PDA) plates were used to grow C. lunata, R. solani, V. dahilae A. alternata, and A. brassiciola (A. niger was grown on malt extract (ME) agar plates) at 25 °C. When sufficient growth was observed, the mycelia were harvested by punching a hole in the growing fungi and transferring it to a new agar plate. The plate was incubated at 25 °C for 48 h to 72 h to allow formation of a radial mycelial colony. 6 mm discs impregnated with 20 μL of mO1 were placed equidistant (1 cm) from the growing ends of the mycelia and incubated for 24 h at 25 °C. Deionized water was used as a negative control. Formation of arc-shaped inhibition zones indicated susceptibility to mO1.
Microbroth dilution assay
A half maximal inhibitory concentration (IC50) of mO1 against A. alternata and A. brassiciola was obtained by performing a microbroth dilution assay as described by Wiegand et al. . Fungal spores were seeded in half-strength potato dextrose broth at a density of 2.5 × 103 cells/mL. Peptide samples (900 μg/mL to 0.05 μg/mL) were added to the spore suspensions in a 96-well plate and incubated for 24 h at 25 °C. After incubation, the cells were fixed with 100% methanol for 30 min followed by staining with 1% (w/v) methylene blue in 0.01 M borate buffer for 30 min . Excess dye was washed with water, the plates were dried, and stain was eluted with 1% (v/v) ethanol/0.1 N HCl. Absorbance was read at 650 nm using an Infinite@ 200 PRO Tecan microplate reader (Tecan Group Ltd, Germany). Control microcultures contained half-strength potato dextrose broth alone. Percentage growth inhibition was calculated as 100 times the ratio of absorbance of the test microcultures over the absorbance of the control microcultures.
Thermal stability assay
Purified mO1 (40 μg) was dissolved in water and incubated at 100 °C for 1 h. A linear peptide RV-14 (RLYRRGRLYRRNHV) synthesized in our laboratory was used as a control. After incubation, the samples were subjected to RP-UPLC to assess the presence and extent of degradation.
Proteolytic enzyme stability assays
Purified mO1 was incubated at 37 °C with pepsin or carboxypeptidase A in 100 mM sodium citrate (pH 4.5) or 50 mM Tris–HCl with 100 mM NaCl (pH 7.5) at a final peptide to enzyme ratio (w/w) of 20:1 and 50:1, respectively. At each time point (0, 2, 4, 6 h) 50 μL of the sample was injected into the UPLC system, and the mass of peaks collected were analyzed by MALDI-TOF MS. A linear peptide WV-14 (WRLYRGRLYRRNHV) synthesized in-house was used as a control.
The cytotoxic effect of morintide, mO1 (1–100 μM) was tested on Vero cells (5 × 104 cells/ml) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS). Vero cells (ATCC CCL-81) were a kind gift from Dr Liu Ding Xiang (School of Biological Sciences, Nanyang Technological University, Singapore). After 24 h of incubation, 10 μl of PrestoBlue® Cell Viability Reagent (Invitrogen, USA) was added to the wells and incubated at 37 °C for 1 h. After incubation, the absorbance was measured at 570 nm using a microplate reader.
NMR experiments and structure determination
The assignment and structure determination were achieved using 2D 1H-1H TOCSY and NOESY experiments. The spectra were recorded using a Bruker 800 MHz NMR spectrometer (Bruker, IL, USA) equipped with a cryogenic probe. The temperature for the NMR experiments was 298 K. The concentration of mO1 peptide was 1 mM. The lyophilized peptide was dissolved in Milli-Q water, containing 5% D2O (pH approximately 3.5). The mixing times of TOCSY and NOESY experiments were 80 and 200 ms, respectively. The center of the spectrum was 4.735 ppm, and the spectrum width was 12 ppm. The spectra were processed using the software NMRpipe . Assignment of the NOESY spectrum was accomplished using the software Sparky 3.115 . To identify the amide protons involved in hydrogen bonds, an H/D exchange NMR experiment was conducted at 298 K, using a Bruker 600 MHz NMR spectrometer (Bruker, IL, USA) equipped with a cryogenic probe. The lyophilized peptide was dissolved in 100% D2O. 1D NMR spectra were recorded a long time after the peptide dissolved in D2O. In total, 13 spectra were recorded for 12 h. The structure calculation was done using the software CNSsolve 1.3 . The structures were verified by the server PDBsum  to generate a Ramachandran plot. The structure is displayed by the software Chimera 1.6.2 .
Screening and isolation of peptides from M. oleifera
A small-scale screening of M. oleifera leaves revealed a cluster of peptides, with masses of approximately 4 kDa. A scale-up extraction of 3 kg of plant material was performed by mincing in an equal volume of water. Putative cysteine-rich peptides (CRPs) were isolated by multiple rounds of SCX and RP-HPLC. The MS profile of M. oleifera indicated that two major peptide peaks with m/z of 4536.71 Da and 4463.76 Da were present in the leaf extract and were designated morintide mO1 and mO2, respectively. After reduction and alkylation of the disulfide bonds, a mass shift of +474 Da was observed indicating the presence of eight cysteine residues. Sequencing of morintides was performed by nanospray MS/MS. Overall, the sequence contained cysteine (eight residues), glycine (six residues), asparagine (five residues) and glutamine (five residues), making up approximately 50% of the morintide peptide sequence. The glutamine residue at the N-terminus was spontaneously converted to pyroglutamic acid, making the peptide resistant to degradation by exopeptidases.
NMR structure of morintide mO1
Chitin binding activity
To assess the chitin binding activity of morintides, mO1 was incubated with chitin beads for 4 h at 4 °C. RP-UPLC analysis showed that mO1 bound to chitin beads within 1 h (Additional file 6: Figure S2). Using elution buffers with up to 1 M NaCl, mO1 could not be eluted from the beads indicating a strong binding interaction. Use of 0.5 M acetic acid with heating at 100 °C effectively eluted mO1 in 1 h (Additional file 6: Figure S2). These data indicate that mO1 binds strongly to chitin beads, as it only could be eluted using a strong elution buffer and heating at high temperature.
Anti-fungal activity of morintide
Heat and proteolytic stability
The cytotoxic effect of morintide was tested on Vero cells using a PrestoBlue cell viability reagent. No significant cytotoxic effect was observed with concentrations up to 100 μM.
Biosynthesis of morintides
A total of 78,561 transcripts with contig length between 201–22191 bp were assembled from transcriptome of RNA extracted from fresh M. oleifera leaves using Trinity after Illumina HiSeq performed by Macrogen Inc., Korea. These transcripts were analyzed using in-house software, Protein Analyzer 1.0 to look for conserved cysteine motifs. Two precursor sequences that were designated mo1 and mo2 were obtained. The precursors mo1 and mo2 comprise a 20-amino-acid-long ER signal peptide followed by a 44-amino acid long mature peptide domain and a short 15 amino acid-long C-terminal domain. Sequence comparison of mo1 and mo2 revealed that the signal peptide is identical, the mature domain differs in one amino acid residue, and the C-terminal domain varies at three positions, specifically, Asp-67, Glu-71 and Gly-76 of mo1 are replaced by Ala-67, Gly-71 and Ser-76 in mo2. The precursor organization of hevein-like peptides differs from other cysteine-rich peptides that contain an N-terminal pro-domain before the mature peptide domain [30, 49, 50] and suggests that morintides are ribosomally synthesized peptides  whose bioprocessing occurs through the secretory pathway .
In this study, two novel 8C-hevein-like peptides, collectively named morintides, were isolated, sequenced and characterized The 44 amino-acid-long morintides, were detected at proteomic and transcriptomic levels and differ by one amino acid residue. Morintides are homologous to hevein and other 8C-hevein-like peptides such as Pn-AMPs  and Fa-AMPs . Structural analysis of mO1 by NMR revealed that three disulfide bonds were arranged to form the cystine knot motif, and the fourth disulfide bond was located at the C-terminus. Morintide mO1 inhibited the growth of A. alternata and A. brassiciola in the disc diffusion assay with an IC50 in the micromolar range. Transcriptomic analysis revealed that the overall precursor organization of morintides is similar to hevein-like peptides except for their short, protein-cargo-free C-terminal domain.
Sequence comparison of morintides
Sequence comparison of morintides with hevein and reported 8C-hevein-like peptides revealed that, in addition to the eight conserved cysteine residues, three glutamine (Gln-6, Gln-20, Gln-36), two serine (Ser-19, Ser-37) and Leu-16 were absolutely conserved in all 8C-hevein-like peptides (Table 1). Based on the cysteine-spacing pattern, the peptide sequences can be divided into six intercysteinyl loops. The length of the loops is conserved, with variation observed only in loop 5 of Fa-AMPs and Pn-AMPs that comprises three residues as opposed to the five residues seen in hevein and mO1. The N-terminus of all 8C-hevein-like peptides, except Fa-AMPs, contains a glutamate residue that is spontaneously converted to pyroglutamic acid. The C-terminus usually consists of a positively charged residue followed by glycine, serine or aspartate residues in mO1, Pn-AMP1 and hevein, respectively. Morintides displayed the least sequence identity with the proline-rich ginkgotides, probably because they are isolated from a gymnosperm, G. biloba.
The overall structure of CRPs is stabilized primarily by the disulfide-rich core along with the secondary structure and hydrophobic contacts . The disulfide core of hevein-like peptides has a knotted topology characterized by two adjacent disulfide bonds that are crossed-over by a third disulfide bond resulting in the cystine-knot confirmation . Consequently hevein-like peptides are highly stable to degradation by heat and enzymes. Morintide, mO1 has a similar disulfide arrangement. The secondary structure of hevein-like peptides, including hevein itself , Ac-AMP2 from Amaranthus caudatus , aSG1 from Alternanthera sessilis , WAMP-1a from Triticum kiharae  and Ee-CBP from Euonymus europaeus , comprises at least two β-strands and a short \( \alpha \)-helical turn. The secondary structure of mO1 is similar to the reported hevein-like peptides and comprises two anti-parallel β-sheets but lacks the \( \alpha \)-helical turn at the C-terminus. The C-terminal residue of mO1 is Arg, as opposed to Pro-33, which is found in hevein. This replacement could result in the absence of the \( \alpha \)-helical turn in mO1. The chitin-binding property of hevein-like peptides is determined by the residues occupying the chitin-binding site located close to the knottin core. In hevein, binding interactions with the N-acetylglucosamine residues are stabilized by CH-π stacking interactions and van der Waal’s contacts involving Trp-21, Trp-23 and Tyr-30 . A hydrogen bond between the sugar moiety and the hydroxyl group of Ser-19 stabilizes the complex. Due to sequence homology of hevein and mO1 it is presumed that the chitin-binding interaction is through a similar interaction in mO1.
Anti-fungal activity of morintides
Of the seven fungal strains tested, the morintide mO1 could inhibit the growth of A. alternata and A. brassiciola, as indicated by the arc-shaped inhibition zones in the disc diffusion assay. Morphological changes, such as swollen hyphal tips, stunted growth and less branched hyphae, were observed after treatment of A. alternata and A. brassiciola with different concentrations of mO1. Similar changes in morphology have been observed with hevein , IWF-4  and Ee-CBP [58, 61]. At present, there are two proposed mechanisms by which hevein-like peptides may inhibit fungal growth. First, the chitin-binding property of hevein-like peptides might cause them to bind to nascent chitin fibers in newly formed chitin chains of growing hyphae, disrupt cell wall morphogenesis and impede fungal growth . Second, the small size and highly basic PI of hevein-like peptides might allow them to easily penetrate through the fungal cell wall, reach the plasma membrane, and alter membrane polarity, causing leakage of cytoplasmic material and subsequently inhibiting fungal growth [9, 58, 61]. The mechanism of action of morintides remains to be elucidated; however, the anti-fungal activity of morintides can be attributed to their chitin-binding property as they have neutral PI values.
Biosynthesis of morintides
Transcriptome data analysis revealed that morintides are expressed as three-domain precursors and are processed via the secretory pathway . The N-terminal cleavage site of the signal peptidase is a highly conserved Ala residue in morintides, while C-terminal processing occurs at an absolutely conserved Gly residue (Additional file 7). The 15-amino-acid-long C-terminal domain of morintides is rich in glycine residues and differs from hevein and 10C-hevein-like peptides, which have a significantly longer C-terminal domain (142–200 amino acid residues) carrying a bio-active protein cargo with a Barwin domain or a glycine or serine-rich hinge region followed by the catalytic domain of class-I chitinases [62, 63]. To determine whether the C-terminal domain of mo1 plays a role as a targeting signal in directing the mature peptide to vacuoles, a blastp search was performed in the NCBI database. The search result shows no homology to any known proteins in the NCBI database. Thus the role of the C-tail in morintides remains to be determined.
The glycine-rich nature of the C-terminal domain of morintides suggests that it could be a remnant of the hinge-region in chitinases, and morintides could be truncated chitinases. The precursor organization of hevein-like peptides is similar to ginkgotides  and thionins  but differs from other CRPs such as cyclotides, which contain an N-terminal pro-peptide before the mature peptide [31, 36, 49, 65].
Evolution and origin of morintides
The hevein-like peptide family can be divided into three sub-classes, making it an interesting family to study genetic diversity. Genetic divergence within the plant phyla results from mutations in the mature peptide, which also lead to functional diversification. For example, the signal peptide and C-terminal domain of morintide mO1 and mO2 precursors are highly homologous, but a point mutation in the mature domain results in replacement of Gln-15 in mO1 to Gly-15 in mO2. Similar genetic diversity has also been observed in other CRPs such as cliotides cT4-cT12 of the cyclotide family isolated from Clitoria ternatea , allotides of the cystine knot α-amylase family isolated from Allamanda cathartica  and α- and β-hordothionin of the thionin family isolated from Hordeum vulgare .
In this study, two novel 8C-hevein-like peptides (mO1 and mO2), collectively named morintides, were isolated and characterized from the leaves of M. oleifera. NMR studies of the 44-amino-acid-long morintides revealed that they contain four disulfide bonds. The highly disulfide-constrained structure renders mO1 resistant to thermal and enzymatic degradation, making it an interesting scaffold for drug development. Morintide, mO1 can inhibit the growth of A. alternata and A. brassiciola with IC50 in the micromolar range. It caused morphological changes such as swollen hyphal tips with stunted and less-branched hyphae. Transcriptome data analysis revealed that morintide precursors comprise a signal peptide domain, a mature peptide domain and a C-terminal domain. Comparison of morintide precursors with other hevein-like peptides showed that they contain a radically shortened C-terminal domain, which could be the result of mRNA splicing or frame shift deletions in large chitinase precursors. This stark difference in the length of the C-terminal domain is an interesting finding that sheds light on the evolution of hevein-like peptides and helps define a novel sub-group of 8C-hevein-like peptides. Together, this work expands the library of 8C-hevein-like peptides and sheds light on their evolutionary relationship and biosynthetic pathway. We have also gained insights into the chitin-binding property and the fungal growth inhibition of morintides.
High-performance liquid chromatography
- MALDI-TOF MS:
Matrix-assisted laser desorption/ionization-time of flight mass spectrometry
Nuclear Overhauser effect spectroscopy
Polymerase chain reaction
Strong cation exchange
Ultra-performance liquid chromatography
This research was supported by a Competitive Research Grant from the National Research Foundation in Singapore (NRF-CRP8-2011-05). We thank Dr. Aida Serra from Prof. Newman Sze’s laboratory at NTU for her help with LC-ESI MS/MS.
This research was supported by a Competitive Research Grant from the National Research Foundation in Singapore (NRF-CRP8-2011-05) and an NTU iFood Research Grant (M4081467.080).
Availability of data and materials
The NMR structure of mO1 can be viewed with PDB accession code 5WUZ and BMRB code 36040. The complete mRNA coding sequence of mO1 and mO2 can be found in GenBank with the accession codes KY436355 and KY436356, respectively. The phylogenetic tree, sequence data and alignments have been deposited on TreeBASE database and can be accessed via the permanent link: http://purl.org/phylo/treebase/phylows/study/TB2:S20670.
JPT, SGK, KHW and WLT conceived and designed the experiments. SGK, KHW, WLT and TX performed the experiments, analyzed the data and wrote the manuscript. JPT revised the manuscript. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
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